EUS-guided CTCs + Multi-omics: Predicting Pancreatic Cancer Recurrence and Metastases

Exploratory Study on EUS-guided Portal Vein CTCs and Their Subtypes Combined With Multi-omics Detection for Early Warning of Pancreatic Cancer Recurrence and Metastasis

The investigators conduct a single-center, prospective, observational study to explore the value of EUS-guided portal vein circulating tumor cells (PV-CTCs) and their subtypes combined with multi-omics tests in the early warning of recurrence and metastasis of resectable pancreatic cancer(RPC) and borderline resectable pancreatic cancer (BRPC).

A total of 20 participants are stratified based on imaging studies into the resectable pancreatic cancer group (8 cases), the borderline resectable pancreatic cancer group (8 cases), and the locally advanced pancreatic cancer group (4 cases). These patients have solid space-occupying lesions (with a diameter >1cm) within the accessible range of endoscopic ultrasound (EUS) for the pancreas as indicated by imaging studies. Every trial patient will get a unique identification number, and it will not change through out the whole trial.

Use the inclusion and exclusion criteria to observe the patients and do relative inspections, and confirm if the patients qualified or not to the trial. Record the result of last time test before the treatment. Although it is better to get the informed consent before doing all kinds of observation and tests, if for some reason, the medical imaging examination has completed, as long as the imaging examination was done within 3 weeks before the needle biopsy, it can still be collect as baseline data; other lab test items done at 2 weeks before the needle biopsy can still be collect as baseline data for pre-research use, but these tests should be done at the trail center hospital so as to guarantee the data trace ability.

The investigators will do the sampling procedures for all of them in a strict sequence: first, under EUS guidance, 10ml of portal vein blood is prioritized for collection using a 22G ECHO 3-22 needle to avoid contamination. Subsequently, the investigators will do the needle passes for 3 times on the pancreatic lesion with 5 mL wet suction for tissue acquisition. Finally, 10ml of peripheral blood is collected. Samples are processed using CTC100 system or IFC-IMP technology, followed by scRNA-seq (10xGenomics) and proteomic analysis (timstofPro2/orbitrap). The results will be fed back to clinicians within 3 working days. Without knowing the sample source (blinded to specific subtypes initially), the cytologist and pathologist evaluate the specimen quality and make diagnosis.

Follow up (outpatient follow up or telephone follow up) the patients at 1 week, 12 weeks and 36 weeks after the puncture and collect the patients clinical data and confirm their final diagnosis.

During the trial, if severe adverse event occurs, the trialed center must take immediate actions necessary to guarantee the trialed patients' safety. Once severe adverse event occurs, the researchers should inform the trial applicant and the trail center's ethics committee within 24 hours after the researchers gets to know the adverse event. And the researchers should also fax the report to State Food and Drug Administration of China and the local provincial food and drug administration. After receiving the report, the applicant should inform other clinical trial centers within 24 hours. All the severe adverse events should be filed at group leader medical center and other trial centers.

Case Report Form (CRF ) will be filled by the researchers, every involved patient must have the CRF filled. This will be audited by clinical monitor and handed over to data administrator to input and manage data, the first copy will be kept by the applicant, the second copy will go to the trial center, and the third copy will be kept by the trail researchers. The data input and management will be taken care by specially assigned person. In order to guarantee the data accuracy, data input will be done twice by two independent data administrators, by computerized and manual verifying, hand over the data to statistical experts to do check and statistic analyzing.

For the questions and doubts within the case report form, the data administrator make Data Resolution Query (DRQ) and via the clinical monitor asking the researchers. The researchers will answer and feed back as soon as possible. According to the researchers answer, data administrator will do the data modifying, confirming or inputting, and when necessary send out DRQ again.

This will be done by specialized statistic analyzing people according to the predetermined statistic analyzing plan. The statistic analyze will be carried out according to intention principle confirmed full analysis set and per-protocol set principle. After completing the statistic analyzing, the statistic analyzer issue the statistic analysis report and send this to major researchers to write the study report.

Statistic analyzing plan: ⑴ General principle: ① all the statistic tests are use the two-tailed-test method, P<0.05 will be thought as the tested difference is statistical significance. ② the quantitative indicator description will calculate the Mean and Standard deviation. The classification indicator description will describe the cases and percentage of all types of cases. ⑵ Statistic analyzing method: ① for the measurement data, compare the difference between Portal Vein CTCs and Peripheral Blood CTCs, use paired t-test or symbol rank sum test to compare with the difference within the group. ② for the counting data (CTC subtypes), use x2 test or Fisher's exact test method to compare the groups. ⑶ Shedding analysis: Comparison of groups' total shedding rates and the shedding rates caused by adverse events will use x2 test or Fisher's exact test method. ⑷ The baseline value's equilibrium analysis: Use group t test or x2 test to compare the demography info and vital signs, disease history, and basic treatment and other indicators of baseline value, so as to measure the balance of the groups. ⑸ Effectiveness analysis: The major indicator of effectiveness analysis is the correlation between PV-CTC quantity/subtypes and the recurrence/metastasis of BRPC, and the indicators of second effectiveness include the applicability of neoadjuvant therapy guidance. ⑹ Safety analysis: Use x2 test or Fisher's exact test to compare the adverse event/adverse reaction (include puncture complications like pancreatitis, bleeding) rates. And use table to describe the adverse events during this trial project; the lab test results before and after the trial, the normal/abnormal changing condition and the relationship with this trial research when abnormal changes happened.

Inclusion Criteria:

1. Patients with solid masses (diameter > 1 cm) in the pancreatic area within the accessible range of endoscopic ultrasound, as indicated by clinical symptoms, laboratory tests, and imaging examinations (MRI, CT, B-ultrasound), who require biopsy to clarify the nature of the lesion.
2. Patients diagnosed with resectable pancreatic cancer, borderline resectable pancreatic cancer, locally advanced pancreatic cancer by imaging(CT/MRI).
3. Newly diagnosed pancreatic cancer patients who have not received radiotherapy or chemotherapy.
4. Signed informed consent form.
5. Patients must be able to comply with the trial requirements.

Exclusion Criteria:

1. Patients with other active malignant tumors
2. Patients with coagulation dysfunction (PLT 50,000/mm3, INR > 1.5; roughly estimated, INR > 1.5 is approximately equivalent to PT > 18 seconds)
3. Pregnant women
4. Patients with hemorrhagic diseases
5. Patients with a history of taking anticoagulant drugs such as aspirin and warfarin in the past week
6. Patients with absolute contraindications to EUS examination, a history of acute pancreatitis within the past 2 weeks, a history of gastric surgery, pregnancy, severe diseases, or a history of allergy to anesthetics
7. Patients whose EUS examination was terminated early due to esophageal stenosis, obstruction, large space-occupying lesions, rapid changes in the patient's heart rate or respiratory rate, patient intolerance, or a large amount of food residue, etc.
8. Patients with known hepatitis C virus infection
9. Patients with known human immunodeficiency virus (HIV) infection
10. Patients with imaging examinations, EUS results, etc., suggesting pancreatic cystic space-occupying lesions
11. Patients who have received radiotherapy or chemotherapy
12. Patients with incomplete pathological information, unable to make a clear diagnosis, or unable to sign the informed consent form